1122
BIOCHEMICAL SOCIETY TRANSACTIONS
difficult to rationalize on the basis of a homogeneous pool of quinones. A more conceivable model is one that postulates the presence of separate quinone pools or domains as the electron acceptors for the dehydrogenases (see Gutman, 1977; Rich & Bonner, 1978). In such a model it is proposed that DBCT interacts with a pool or domain of quinone common to succinate and endogenous NADH dehydrogenases. In conclusion our experimental results strongly indicate that DBCT is a potent electron-transport inhibitor localized on the substrate side of the ubiquinone-cytochrome b region of the respiratory chain. This work was partly supported by a Rank Prize Research Grant. Cain, K., Partis, M.D. & Griffiths, D. E. (1977) Biochem. J. 166,593-602 DeChadarevjan, S., DeSantis, A., Melandri, B. A. & Baccarini-Melandri, A. (1979) FEBS Lett. 97,293-295’ Douce, R., Moore, A. L. & Neuberger, M. (1977) Plant Physiol. 60,625-628 Griffiths, D. E. (1976) Biochem. J. 160,809-812 ) Lett. 74,3841 Griffiths, D. E., Hyams, R. L. & Bertoli, E. ( 1 9 7 7 ~FEBS Griffiths, D. E., Hyams, R. L. & Partis, M. D. (1977b) FEBS Letf.78, 155-160 Gutman, M. (1977) in Bioenergefics ofMembranes (Packer, L., Pagageorgiou, G. C. & Trebst, A., eds.), pp. 165-175, Elsevier/North Holland Partis, M. D., Hyams, R. L. & Griffiths, D. E. (1977) FEBS Lett. 75,47-51 Rich, P. R. & Bonner, W. D., Jr. (1978) Biochim. Biophys. Acta 501, 381-395 Singh, A. P. & Bragg, P. D. (1978) Biochem. Biophys. Res. Commun. 81,161-167
Photorespiratory Nitrogen Cycling :Evidence for a Mitochondria1 Glutamine Synthetase CHRISTOPHER JACKSON, JANE E. DENCH, PHILLIP MORRIS, SEUNG C. LUI, DAVID 0. HALL and ANTHONY L. MOORE* University of London King’s College, Department of Plant Sciences, 68 Ha[fMoonLane, London SE24 9JF, U.K.
The photorespiratory efflux of CO, from the leaves of C3 plants has received considerable attention (Tolbert, 1971 ;Zelitch, 1975; Halliwell, 1978). Recently, the development of a successful technique for the isolation of intact mitochondria from leaf tissues (Douce et al., 1977) has allowed a more detailed investigation of the mitochondria1 glycine decarboxylase, which is currently considered to be the major C0,-releasing system during photorespiration (Tolbert, 1971; Halliwell, 1978). In comparison, little emphasis has so far been placed on the fate of NH3, which is released stoicheiometrically and simultaneously with the COz (cf. Miflin & Lea, 1977). Various techniques are available for the isolation of organelles of high integrity from the leaves of higher plants. However, such preparations are contaminated with other subcellular structures. Table 1 shows the distribution of various enzymes during the subcellular fractionation of spinach leaf (Spinaciu oleracea L.) homogenates (Jackson e t al., 1979) expressed as the percentage of the activity present in the original homogenate. Although the bulk of the glutamine synthetase (measured by the coupled assay of Shapiro & Stadtman, 1970) was recovered in the soluble fraction, the distribution was very similar to that of NADP-glyceraldehyde 3-phosphate dehydrogenase (Bradbeer, 1969), a known chloroplast stromal enzyme (Halliwell, 1978). The results suggest that most of the glutamine synthetase activity is released from the chloroplast during the fractionation procedure. About 3 % of the synthetase activity was associated with the washed mitochondria, which contained 44 % of the cytochrome c oxidase (measured according to Tolbert,
* Present address: Department of
Biochemistry, University of Sussex, Falmer, Brighton,
U.K.
1979
1123
583rd MEETING, CAMBRIDGE Table 1. Subcellular fractionation of spinach leaves
Recovery is expressed as % of the total activity present in the initial homogenate. Glutamine synthetase was measured after treatment with Triton X-100 (0.045 % final concentration). Specific activities (nmol/min per mg of protein) are shown in parentheses. Fractions were prepared by the method of Jackson et al. (1979) except gradient mitochondria (Jackson & Moore, 1979). Fraction 3000g pellet Crude chloroplast fraction
Chlorophyll
TPDH*
90
18 (15)
12000g pellet Crude mitochondrial fraction
7
12000g supernatant Soluble fraction 4 11000g pellet Washed mitochondrial fraction 2.5 Gradient mitochondria
0.06
Cytochrome Glutamine synthetase c oxidase 22 (32.6) 7 (73.4) 70 (24.8) 3 (34.7) 1.5 (71.8)
* NADP-glyceraldehyde 3-phosphatedehydrogenase. 1974). Thus not more than 7-8 % of the total leaf glutamine synthetase could be contained in the mitochondria. The specific activity of this enzyme in the washed mitochondria was about 35 nmol/min per mg of mitochondrial protein, compared with about 26nmol/min per mg of total protein for the remainder. In consideration of these results, if a glutamine synthetase is present in the cytosol, it can only represent a very small proportion of the total activity in the leaf. The subcellular distribution of glutamine synthetase would therefore seem to be similar to that for superoxide dismutase (Jackson et al., 1978). Since the washed mitochondrial fractions are contaminated with broken chloroplasts, the observed activities of glutamine synthetase in the mitochondria could be due to this contamination. Purification of the mitochondria by silica sol gradient centrifugation (Jackson & Moore, 1979) indicates that this is not the case. Mitochondria1 preparations obtained by this procedure show high integrity and ADP :0 ratios, and were found t o retain the glutamine synthetase activity originally present in the washed fractions (Table 1). The specific activity of the synthetase was increased to about 72nmol/ min per mg of mitochondrial protein after gradient separation. This glutamine synthetase activity would be sufficient to account for the photorespiratory release of NH3 from glycine decarboxylase (Moore et al., 1977) (specific activity approximately 70nmol/min per mg of mitochondrial protein). The increase in specific activity of these enzymes is comparable with that obtained for cytochrome c oxidase (2-4-fold) with similar gradients. Glutamine synthetase activity was slightly increased after osmotic swelling of the mitochondria. However, the maximum synthetase activity could be obtained only by treatment with detergent such as Triton X-100 (final concentration 0.045 %). This would indicate a close association with the inner mitochondrial membrane. These results, in contrast to those of Keys et al. (1978), suggest the presence of a mitochondrial glutamine synthetase. Consequently the NH3 released by glycine decarboxylase during photorespiration could be rapidly reassimilated into glutamine in the mitochondrion, instead of by an external glutamine synthetase. The glutamine thus produced would then be freely available for use in nitrogen metabolism (see Tate & Meister, 1973).
Vol. 7
1124
BIOCHEMICAL SOCIETY TRANSACTIONS
This work was supported by a grant from the Rank Prize Research Funds. Bradbeer, J. W. (1979) New Phytol. 68,233-245 Douce, R., Moore, A. L. & Neuburger, M. (1977) Plant Physiol. 60,625-628 Halliwell, B. (1978) Prog. Biophys. Mol. Biol. 33, 1-54 Jackson, C., Dench, J. E., Moore, A. L., Halliwell, B. & Hall, D. 0. (1978) Eur. J. Biochem. 91,339-344
Jackson, C. & Moore, A. L. (1979) in Methodological Surveys in Biochemistry (Reid, E., ed.) vol. 9, Plant Organelles, Ellis Horwood, Chichester, U.K. in the press Jackson, C., Dench, J.E., Hall, D. 0. & Moore, A. L. (1979) Plant Physiol. in the press Keys, A. J., Bird, I. F., Cornelius, M. J., Lea, P. J., Wallsgrove, R. M. & Miflin, B. J. (1978) Nature (London) 275,741-743
Miflin, B. J. & Lea, P. J. (1977) Annu. Rev. Plant Physiol. 28,299-329 Moore, A. L., Jackson, C., Halliwell, B., Dench, J. E. & Hall, D. 0. (1977) Biochem. Biophys. Res. Commun. 78,483-491
Shapiro, B. M. & Stadtman, E. R. (1970) Methods Enzymol. 129,910-922 Tate, S . S. & Meister, A. (1973) in The Enzymes of GIutamine Metabolism (Prusiner, S . & Stadtman, E. R., eds.), pp. 77-127, Academic Press, New York Tolbert, N. E. (1971) Annu. Rev. Plant. Physiol. 22,45-74 Tolbert, N. E. (1974) Methods Enzymol. 31A, 734-746 Zelitch, I. (1975) Annu. Rev. Biochem. 44,123-145
The Effect of Lead Nitrate on Purified Pig Heart Pyruvate Dehydrogenase KEITH HYLAND and DAVID C. H. McBRIEN Department of Biochemistry, Brunel University, Uxbridge, Middx. UB8 3PH, U.K.
Ulmer & Vallee (1969) have shown that lipoamide dehydrogenase, a part of the pyruvate dehydrogenase complex (EC 1.2.4.1) is inhibited by lead at concentrations less than 1 0 - 5 ~Their . work did not yield information on the effect of lead on the whole of the pyruvate dehydrogenase complex. The pyruvate dehydrogenase complex is a multicomponent assembly consisting of three sets of enzymes which by oxidative degradation of pyruvate lead to the formation of acetyl-CoA. The three enzymes are pyruvate decarboxylase, dihydrolipoate acetyltransferase and dihydrolipoate dehydrogenase (lipoamide dehydrogenase). Reaction mechanisms are well known (Gunsalus, 1954; Reed, 1960; Ullrich & Mannschreck, 1967). Associated with the three main enzymes of the complex are two regulatory enzymes: pyruvate dehydrogenase phosphate phosphatase and pyruvate dehydrogenase kinase (Linn et al., 1969). The kinase requires ATP and inactivates the complex by phosphorylation of certain serine groups on pyruvate decarboxylase (Linn et al., 1972). Reactivation occurs by removal of the phosphate by the pyruvate dehydrogenase phosphate phosphatase. Analysis of the pyruvate dehydrogenase complex has been studied in Reed’s laboratory (Linn et al., 1972; Reed, 1974; Roche & Reed, 1972). In bovine heart the complex contains approximately 60 transacetylase molecules, 30 pyruvate decarboxylase molecules and 12 dihydrolipoyl dehydrogenase molecules. Associated with these are five phosphatase molecules and five kinase molecules. Inhibition of pyruvate dehydrogenase by lead may thus arise from its action on the main enzyme complex or by its action on one of the regulatory enzymes. We have investigated the effects of lead on the activity of the main enzyme complex and upon the two regulatory enzymes. Fully activated pyruvate dehydrogenase, low in ATPase activity, was prepared from pig heart by the method of Cooper et al. (1974). Pyruvate dehydrogenase phosphate phosphatase was prepared by the method of Denton et al. (1972). Before use both preparations were dialysed at 5°C against several litres of 20m~-Tris/HC1,pH 7.0, containing 5 mM-mercaptoethanol in order to remove the phosphate ions. 1979
BIOCHEMICAL SOCIETY TRANSACTIONS
difficult to rationalize on the basis of a homogeneous pool of quinones. A more conceivable model is one that postulates the presence of separate quinone pools or domains as the electron acceptors for the dehydrogenases (see Gutman, 1977; Rich & Bonner, 1978). In such a model it is proposed that DBCT interacts with a pool or domain of quinone common to succinate and endogenous NADH dehydrogenases. In conclusion our experimental results strongly indicate that DBCT is a potent electron-transport inhibitor localized on the substrate side of the ubiquinone-cytochrome b region of the respiratory chain. This work was partly supported by a Rank Prize Research Grant. Cain, K., Partis, M.D. & Griffiths, D. E. (1977) Biochem. J. 166,593-602 DeChadarevjan, S., DeSantis, A., Melandri, B. A. & Baccarini-Melandri, A. (1979) FEBS Lett. 97,293-295’ Douce, R., Moore, A. L. & Neuberger, M. (1977) Plant Physiol. 60,625-628 Griffiths, D. E. (1976) Biochem. J. 160,809-812 ) Lett. 74,3841 Griffiths, D. E., Hyams, R. L. & Bertoli, E. ( 1 9 7 7 ~FEBS Griffiths, D. E., Hyams, R. L. & Partis, M. D. (1977b) FEBS Letf.78, 155-160 Gutman, M. (1977) in Bioenergefics ofMembranes (Packer, L., Pagageorgiou, G. C. & Trebst, A., eds.), pp. 165-175, Elsevier/North Holland Partis, M. D., Hyams, R. L. & Griffiths, D. E. (1977) FEBS Lett. 75,47-51 Rich, P. R. & Bonner, W. D., Jr. (1978) Biochim. Biophys. Acta 501, 381-395 Singh, A. P. & Bragg, P. D. (1978) Biochem. Biophys. Res. Commun. 81,161-167
Photorespiratory Nitrogen Cycling :Evidence for a Mitochondria1 Glutamine Synthetase CHRISTOPHER JACKSON, JANE E. DENCH, PHILLIP MORRIS, SEUNG C. LUI, DAVID 0. HALL and ANTHONY L. MOORE* University of London King’s College, Department of Plant Sciences, 68 Ha[fMoonLane, London SE24 9JF, U.K.
The photorespiratory efflux of CO, from the leaves of C3 plants has received considerable attention (Tolbert, 1971 ;Zelitch, 1975; Halliwell, 1978). Recently, the development of a successful technique for the isolation of intact mitochondria from leaf tissues (Douce et al., 1977) has allowed a more detailed investigation of the mitochondria1 glycine decarboxylase, which is currently considered to be the major C0,-releasing system during photorespiration (Tolbert, 1971; Halliwell, 1978). In comparison, little emphasis has so far been placed on the fate of NH3, which is released stoicheiometrically and simultaneously with the COz (cf. Miflin & Lea, 1977). Various techniques are available for the isolation of organelles of high integrity from the leaves of higher plants. However, such preparations are contaminated with other subcellular structures. Table 1 shows the distribution of various enzymes during the subcellular fractionation of spinach leaf (Spinaciu oleracea L.) homogenates (Jackson e t al., 1979) expressed as the percentage of the activity present in the original homogenate. Although the bulk of the glutamine synthetase (measured by the coupled assay of Shapiro & Stadtman, 1970) was recovered in the soluble fraction, the distribution was very similar to that of NADP-glyceraldehyde 3-phosphate dehydrogenase (Bradbeer, 1969), a known chloroplast stromal enzyme (Halliwell, 1978). The results suggest that most of the glutamine synthetase activity is released from the chloroplast during the fractionation procedure. About 3 % of the synthetase activity was associated with the washed mitochondria, which contained 44 % of the cytochrome c oxidase (measured according to Tolbert,
* Present address: Department of
Biochemistry, University of Sussex, Falmer, Brighton,
U.K.
1979
1123
583rd MEETING, CAMBRIDGE Table 1. Subcellular fractionation of spinach leaves
Recovery is expressed as % of the total activity present in the initial homogenate. Glutamine synthetase was measured after treatment with Triton X-100 (0.045 % final concentration). Specific activities (nmol/min per mg of protein) are shown in parentheses. Fractions were prepared by the method of Jackson et al. (1979) except gradient mitochondria (Jackson & Moore, 1979). Fraction 3000g pellet Crude chloroplast fraction
Chlorophyll
TPDH*
90
18 (15)
12000g pellet Crude mitochondrial fraction
7
12000g supernatant Soluble fraction 4 11000g pellet Washed mitochondrial fraction 2.5 Gradient mitochondria
0.06
Cytochrome Glutamine synthetase c oxidase 22 (32.6) 7 (73.4) 70 (24.8) 3 (34.7) 1.5 (71.8)
* NADP-glyceraldehyde 3-phosphatedehydrogenase. 1974). Thus not more than 7-8 % of the total leaf glutamine synthetase could be contained in the mitochondria. The specific activity of this enzyme in the washed mitochondria was about 35 nmol/min per mg of mitochondrial protein, compared with about 26nmol/min per mg of total protein for the remainder. In consideration of these results, if a glutamine synthetase is present in the cytosol, it can only represent a very small proportion of the total activity in the leaf. The subcellular distribution of glutamine synthetase would therefore seem to be similar to that for superoxide dismutase (Jackson et al., 1978). Since the washed mitochondrial fractions are contaminated with broken chloroplasts, the observed activities of glutamine synthetase in the mitochondria could be due to this contamination. Purification of the mitochondria by silica sol gradient centrifugation (Jackson & Moore, 1979) indicates that this is not the case. Mitochondria1 preparations obtained by this procedure show high integrity and ADP :0 ratios, and were found t o retain the glutamine synthetase activity originally present in the washed fractions (Table 1). The specific activity of the synthetase was increased to about 72nmol/ min per mg of mitochondrial protein after gradient separation. This glutamine synthetase activity would be sufficient to account for the photorespiratory release of NH3 from glycine decarboxylase (Moore et al., 1977) (specific activity approximately 70nmol/min per mg of mitochondrial protein). The increase in specific activity of these enzymes is comparable with that obtained for cytochrome c oxidase (2-4-fold) with similar gradients. Glutamine synthetase activity was slightly increased after osmotic swelling of the mitochondria. However, the maximum synthetase activity could be obtained only by treatment with detergent such as Triton X-100 (final concentration 0.045 %). This would indicate a close association with the inner mitochondrial membrane. These results, in contrast to those of Keys et al. (1978), suggest the presence of a mitochondrial glutamine synthetase. Consequently the NH3 released by glycine decarboxylase during photorespiration could be rapidly reassimilated into glutamine in the mitochondrion, instead of by an external glutamine synthetase. The glutamine thus produced would then be freely available for use in nitrogen metabolism (see Tate & Meister, 1973).
Vol. 7
1124
BIOCHEMICAL SOCIETY TRANSACTIONS
This work was supported by a grant from the Rank Prize Research Funds. Bradbeer, J. W. (1979) New Phytol. 68,233-245 Douce, R., Moore, A. L. & Neuburger, M. (1977) Plant Physiol. 60,625-628 Halliwell, B. (1978) Prog. Biophys. Mol. Biol. 33, 1-54 Jackson, C., Dench, J. E., Moore, A. L., Halliwell, B. & Hall, D. 0. (1978) Eur. J. Biochem. 91,339-344
Jackson, C. & Moore, A. L. (1979) in Methodological Surveys in Biochemistry (Reid, E., ed.) vol. 9, Plant Organelles, Ellis Horwood, Chichester, U.K. in the press Jackson, C., Dench, J.E., Hall, D. 0. & Moore, A. L. (1979) Plant Physiol. in the press Keys, A. J., Bird, I. F., Cornelius, M. J., Lea, P. J., Wallsgrove, R. M. & Miflin, B. J. (1978) Nature (London) 275,741-743
Miflin, B. J. & Lea, P. J. (1977) Annu. Rev. Plant Physiol. 28,299-329 Moore, A. L., Jackson, C., Halliwell, B., Dench, J. E. & Hall, D. 0. (1977) Biochem. Biophys. Res. Commun. 78,483-491
Shapiro, B. M. & Stadtman, E. R. (1970) Methods Enzymol. 129,910-922 Tate, S . S. & Meister, A. (1973) in The Enzymes of GIutamine Metabolism (Prusiner, S . & Stadtman, E. R., eds.), pp. 77-127, Academic Press, New York Tolbert, N. E. (1971) Annu. Rev. Plant. Physiol. 22,45-74 Tolbert, N. E. (1974) Methods Enzymol. 31A, 734-746 Zelitch, I. (1975) Annu. Rev. Biochem. 44,123-145
The Effect of Lead Nitrate on Purified Pig Heart Pyruvate Dehydrogenase KEITH HYLAND and DAVID C. H. McBRIEN Department of Biochemistry, Brunel University, Uxbridge, Middx. UB8 3PH, U.K.
Ulmer & Vallee (1969) have shown that lipoamide dehydrogenase, a part of the pyruvate dehydrogenase complex (EC 1.2.4.1) is inhibited by lead at concentrations less than 1 0 - 5 ~Their . work did not yield information on the effect of lead on the whole of the pyruvate dehydrogenase complex. The pyruvate dehydrogenase complex is a multicomponent assembly consisting of three sets of enzymes which by oxidative degradation of pyruvate lead to the formation of acetyl-CoA. The three enzymes are pyruvate decarboxylase, dihydrolipoate acetyltransferase and dihydrolipoate dehydrogenase (lipoamide dehydrogenase). Reaction mechanisms are well known (Gunsalus, 1954; Reed, 1960; Ullrich & Mannschreck, 1967). Associated with the three main enzymes of the complex are two regulatory enzymes: pyruvate dehydrogenase phosphate phosphatase and pyruvate dehydrogenase kinase (Linn et al., 1969). The kinase requires ATP and inactivates the complex by phosphorylation of certain serine groups on pyruvate decarboxylase (Linn et al., 1972). Reactivation occurs by removal of the phosphate by the pyruvate dehydrogenase phosphate phosphatase. Analysis of the pyruvate dehydrogenase complex has been studied in Reed’s laboratory (Linn et al., 1972; Reed, 1974; Roche & Reed, 1972). In bovine heart the complex contains approximately 60 transacetylase molecules, 30 pyruvate decarboxylase molecules and 12 dihydrolipoyl dehydrogenase molecules. Associated with these are five phosphatase molecules and five kinase molecules. Inhibition of pyruvate dehydrogenase by lead may thus arise from its action on the main enzyme complex or by its action on one of the regulatory enzymes. We have investigated the effects of lead on the activity of the main enzyme complex and upon the two regulatory enzymes. Fully activated pyruvate dehydrogenase, low in ATPase activity, was prepared from pig heart by the method of Cooper et al. (1974). Pyruvate dehydrogenase phosphate phosphatase was prepared by the method of Denton et al. (1972). Before use both preparations were dialysed at 5°C against several litres of 20m~-Tris/HC1,pH 7.0, containing 5 mM-mercaptoethanol in order to remove the phosphate ions. 1979
